fimmu-11-00316 February 25, 2020 Time: 19:18 # 1

REVIEWpublished: 27 February 2020

doi: 10.3389/fimmu.2020.00316

Edited by:Ursula Wiedermann,

Medical University of Vienna, Austria

Reviewed by:Arun Kumar,

Coalition for Epidemic PreparednessInnovations (CEPI), Norway

Nitesh K. Kunda,St. John’s University, United States

*Correspondence:Stefan H. E. Kaufmann

Kaufmann@mpiib-berlin.mpg.de

Specialty section:This article was submitted to

Vaccines and Molecular Therapeutics,a section of the journal

Frontiers in Immunology

Received: 13 December 2019Accepted: 07 February 2020Published: 27 February 2020

Citation:Kaufmann SHE (2020)

Vaccination Against Tuberculosis:Revamping BCG by Molecular

Genetics Guided by Immunology.Front. Immunol. 11:316.

doi: 10.3389/fimmu.2020.00316

Vaccination Against Tuberculosis:Revamping BCG by MolecularGenetics Guided by ImmunologyStefan H. E. Kaufmann1,2*

1 Max Planck Institute for Infection Biology, Berlin, Germany, 2 Hagler Institute for Advanced Study, Texas A&M University,College Station, TX, United States

Tuberculosis (TB) remains a major health threat. Although a vaccine has been availablefor almost 100 years termed Bacille Calmette-Guérin (BCG), it is insufficient and bettervaccines are urgently needed. This treatise describes first the basic immunology andpathology of TB with an emphasis on the role of T lymphocytes. Better understandingof the immune response to Mycobacterium tuberculosis (Mtb) serves as blueprint forrational design of TB vaccines. Then, disease epidemiology and the benefits and failuresof BCG vaccination will be presented. Next, types of novel vaccine candidates arebeing discussed. These include: (i) antigen/adjuvant subunit vaccines; (ii) viral vectoredvaccines; and (III) whole cell mycobacterial vaccines which come as live recombinantvaccines or as dead whole cell or multi-component vaccines. Subsequently, the majorendpoints of clinical trials as well as administration schemes are being described. Majorendpoints for clinical trials are prevention of infection (PoI), prevention of disease (PoD),and prevention of recurrence (PoR). Vaccines can be administered either pre-exposureor post-exposure with Mtb. A central part of this treatise is the description of the viableBCG-based vaccine, VPM1002, currently undergoing phase III clinical trial assessment.Finally, new approaches which could facilitate design of refined next generation TBvaccines will be discussed.

Keywords: tuberculosis, vaccine, Bacille Calmette-Guérin, subunit, biomarker, macrophage, T lymphocyte,clinical trial

“ Commit to advancing research for basic science, public health research and the development of innovativeproducts and approaches, . . ., without which ending the tuberculosis epidemic will be impossible, includingtowards delivering, as soon as possible, new, safe, effective, equitable, affordable, available vaccines, . . .”

Resolution adopted by General Assembly of the United Nations from the High Level Meeting on thefight against TB, 2018 (1).

INTRODUCTION

The only tuberculosis (TB) vaccine in use until today, Bacille Calmette Guérin (BCG), wasintroduced in 1921 after intensive research & development (R&D) for more than a decade (2).It was not the first tryout to immunize against TB. The very first attempt was made by RobertKoch who used a subunit-adjuvant formulation (3). Subsequently, several other approaches were

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tested including killed mycobacterial vaccines and live non-tuberculous mycobacterial strains. Yet, these all failed and theonly vaccine with proven safety and efficacy until today remainsBCG. In fact, today BCG is the most widely used vaccine,which has been given more than 4 billion times. BCG wasdeveloped to protect newborns at high risk of TB (2). Thismission has been accomplished at least partially since BCGwas proven to protect against severe extra pulmonary, butless against pulmonary TB in infants (4–6). Yet, even todayinfant TB takes a worrisome toll in TB endemic countries withhigh coverage of BCG immunization (7–9). Later, BCG wasalso tested as a vaccine against pulmonary TB in adolescentsand adults, but this ambitious target was not reached andno vaccine has ever succeeded in reliably protecting againstpulmonary TB, the most prevalent form of the disease, in any agegroup. A better vaccine is urgently needed since Mycobacteriumtuberculosis (Mtb), the cause of TB, remains on top of theinfamous list of deadly infectious agents (10). In 2018, 10 millionindividuals fell ill with this disease and 1.5 million died (11)(Figure 1). The early 21st century has witnessed increasingR&D efforts for novel TB vaccines (12–19). These includesubunit-adjuvant formulations comprising fusion proteins ofMtb, viral vectored vaccines expressing one or more antigensof Mtb, killed mycobacterial vaccines and viable attenuatedmycobacterial vaccines.

IMMUNOPATHOLOGY OFTUBERCULOSIS

Tuberculosis is a chronic infectious disease caused by theintracellular pathogen Mtb (20). This acid-fast bacillus is shieldedby a unique lipoid-rich cell wall containing various wax-likesubstances and glycolipids which contribute to resistance againstimmune attack. Mtb is generally transmitted by aerosols in whichit enters alveoli in lower lung lobes. Once the pathogen has beenengulfed by alveolar phagocytes, it ends up in a phagosome,where it keeps the local pH neutral (21). Moreover, Mtb is capableof egressing into the cytosol (22). These and other mechanismsfacilitate resistance of Mtb to professional phagocytes includingpolymorphonuclear neutrophilic granulocytes (in shortneutrophils) and mononuclear phagocytes (tissue macrophagesand monocytes) (23–27). Resting tissue macrophages generallyfail to eliminate Mtb and serve as its retreat due to their longlifespan. Blood monocytes are slightly more aggressive butfail to achieve sterile elimination of Mtb. Neutrophils arehighly aggressive phagocytes with the potential to harm Mtb.Due to their short lifespan, they generally will not succeed incompletely eliminating Mtb and they do not serve as a harbor,in which Mtb can persist. Once activated by cytokines, notablyInterferon-γ (IFN-γ), mononuclear phagocytes increase theiranti-bacterial capacities and pose a more serious threat to Mtbalthough they generally fail to eradicate it completely. Theinnate immune response mediated by professional phagocytesserves as a first barrier for Mtb. Recent evidence suggests thatepigenetic changes induced by Mtb in professional phagocytesleads to trained immunity. Such trained immunity could play

a role in early defense against repeated Mtb infections (28,29). However, thus far compelling evidence for this notion isstill incomplete.

In addition, subtypes of dendritic cells (DC) can engulf Mtb(30, 31). They likely translocate Mtb into the lung parenchyma,where the formation of a granuloma is initiated.

Granuloma formation is strongly regulated by T lymphocytesoriginally stimulated in the draining lymph nodes to whichDC harboring Mtb navigate (25, 32). T lymphocytes orchestrateformation of solid granulomas which are primarily composedof macrophages, DCs, and T and B lymphocytes. Within thesegranulomas Mtb is contained and the infected individual remainshealthy and develops latent TB infection (LTBI) (24, 33, 34). CD4T cells have been proven to be central to acquired resistanceagainst and containment of Mtb (19, 25). According to thecytokines, these CD4 T cells secrete, they can be categorized intoTH1, TH2, and TH17 cells. TH1 cells are preferentially stimulatedduring Mtb infection and are of major importance for defense.They produce cytokines such as IFN-γ, interleukin-2 (IL-2) andtumor necrosis-α (TNF-α). TH2 cells are only weakly induced.They are often considered harmful in TB since they induceinappropriate effector mechanisms. Their major cytokines areIL-4, IL-5, IL-10, and IL-13. However, evidence has been providedthat TH2 cytokines, at least in part, can contribute to tissuehealing. TH17 cells induce rapid proinflammatory responses bysecreting IL-17. They are stimulated during Mtb infection andevidence has been published that they participate in protectionagainst TB, notably at early stages of infection. The role ofCD8 T cells in protection and containment – although lessprofound – is also widely accepted. CD8 T cells often producecytokines of TH1 type and in addition express cytolytic activity(19, 25, 26). Contribution of cytolytic mechanisms to killing ofMtb has been demonstrated (35). The role of other lymphoidcells including innate lymphoid cells (iLC), NK T cells, mucosaassociated immune T cells (MAIT), γδ T lymphocytes, and Blymphocytes is a matter of ongoing discussion (32, 36–45). Blymphocytes could participate in immunity against TB via twomechanisms: First, as regulatory B lymphocytes and secondas antibody producing plasma cells. Evidence for regulatoryB lymphocytes in immunity against TB is scarce (46, 47).A role for distinct antibody isotypes in defense against TB hasbeen provided (36, 42, 45). Perhaps these antibodies modulateprofessional phagocytes through their binding to distinct Fcreceptors. Convincing evidence has been generated that γδ Tcells contribute to early immune defense by secreting IL-17(38). The iLC can be categorized into iLC-1, iLC-2 and iLC-3according to their cytokine secretion pattern (40). Cytokinesproduced by iLC-1 are of TH1 type, iLC-2 cytokines are ofTH2 and iLC-3 cytokines are of TH17 type. The iLC-1 andiLC-3 probably contribute to resistance to Mtb and the iLC-2to healing of lesions (37). During chronic infection, canonicalCD4 and CD8 T lymphocytes develop into memory T cells whichcan be grouped into effector memory T cells (TEM), centralmemory T cells (TCM), and tissue resident memory T cells(TRM) (48). Although the role of the different memory T cellsin protection against Mtb is incompletely understood, evidencefor a particular role of TRM and TCM in protection against Mtb

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FIGURE 1 | Epidemiologic data for tuberculosis (TB).

has been provided (49, 50). It is likely that different types ofmemory T cells participate in protective immunity at differentstages of infection.

During LTBI, Mtb is contained in solid granuolomas(24, 33, 51). LTBI transforms into active TB disease whengranulomas become necrotic and then caseous. This happensin about 5% of individuals with LTBI within the first 2 yearsand in another 5% at later time points. Thus, only ca. 10%of the 1.7 billion individuals with LTBI develop active TBdisease (52). Progression to active disease is due to weakeningof the immune response via several incompletely understoodmechanisms. It is likely that myeloid-derived suppressor cells andregulatory T lymphocytes participate in dampening of protectiveimmunity (53, 54). These cells produce inhibitory cytokinesincluding IL-4, IL-10, and transforming growth factor-β (TGF-β).Moreover, excessive checkpoint control through inhibitorysurface molecules including PD-1/PDL-1 and CTLA-4/B7co-receptor interactions is likely involved (55, 56).

Notably, progression to active TB from LTBI must be viewedas a continuum rather than a discrete step from one to anotherstage (33, 57, 58). Mtb is transmitted from a TB patient to ahealthy individual in a metabolically active and replicative stage.Hence, the host first encounters highly active Mtb (24). DuringLTBI, Mtb changes from a metabolically active and replicativestage into a dormant stage in which its activities are markedlydownregulated. Once progression to active TB has ensued, Mtbwakes up and becomes active again.

At the early stage of infection, it is possible that Mtb is rapidlyeradicated before stable LTBI develops, but the proportionof individuals who become transiently infected, sometimesaccompanied by a short episode of clinical symptoms remainsunclear (51, 57, 59). Recent evidence suggests LTBI is succeededby incipient TB, in which the host remains healthy, but becomesalerted and Mtb regains its metabolic and replicative activities(59–62). Subsequently, subclinical TB evolves in which firstsigns of pathology occur although clinically the patient appearshealthy. Signs of host vigilance and pathology can be detectedby sensitive gene expression and metabolic profiling (26, 60–62). Given that most, if not all, cases of subclinical TB progressto active TB disease which can be clinically diagnosed, it ispossible to predict disease by sensitive profiling by meansof transcriptomics and metabolomics (60–63). Note that thedifferent stages are not discrete and that in a single patientareas reflecting LTBI (solid granulomas containing dormantMtb), incipient TB (solid to necrotic granulomas in whichMtb regains its metabolic and replicative activity), subclinicalTB (further increase in pathology due to transition of somesolid granulomas to necrotic ones and eventually first signsof caseation) and active TB (all three forms of granulomaspresent with a preponderance toward caseation and cavitation)can coexist. Accordingly, different stages of granulomas rangingfrom solid form to caseation and cavitation coexist, as well (58).Obviously, the coexistence of different pathologies and differentMtb activities render TB immunopathology highly complex.

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BOX 1 | Major vaccine candidates in clinical trials.Different types of TB vaccines have entered the clinical trial pipeline. These are: viral vectored protein antigens of Mtb, fusion protein antigens of Mtb in adjuvants,killed whole mycobacterial cell vaccines, and recombinant viable mycobacterial vaccines. The viral vectored and the adjuvanted protein vaccines are subunitvaccines, which are generally considered to boost a prime with BCG. The viable TB vaccines are considered for BCG replacement or for boosting previous BCGprime. Killed whole cell vaccines are sometimes considered for booster vaccination and more often for TB therapy in adjunct to chemotherapy.

• Viral vectored vaccines include MVA85A, a modified vaccinia Ankara (MVA) vaccine expressing antigen Ag85A of Mtb. First phase IIb efficacy trials withthis vaccine in neonates and in adults failed to provide protection (102, 103). More recently, the vaccine has been tested for safety and immunogenicityafter aerosol application (104, 105). Other viral vectored vaccines include replication deficient adenovirus vectors expressing antigen Ag85A anda replication deficient H1N1 influenza vector expressing antigen Ag85A and ESAT-6. Novel prime boost schedules are also being tested includingadenovirus vectors for prime and MVA vector for boost expressing antigen Ag85A.

Major viral vectored candidates undergoing clinical testing are:Ad5Ag85A (phase I), a replication-deficient adenovirus (Ad) 5 vector expressing Antigen 85A (106, 107).ChAdOx1.85A + MVA85A (phase I), a prime/boost regimen comprising prime with a chimpanzee Adenovirus (ChAd) expressing Antigen 85A(ChAdOx1.85A) followed by a boost with modified Vaccinia Ankara virus (MVA) expressing Antigen 85A (108).TB-FLU-04L (phase IIa), a replication-deficient H1N1 influenza virus strain expressing Antigen 85A and ESAT-6 (109).

• Protein adjuvant formulations undergoing clinical testing include:Hybrid 1 (H1, phase I completed) comprising either IC31 or CAF01 as adjuvant and a fusion protein of Antigen 85B and ESAT-6 as antigen (110, 111).H4 (phase II completed) and H56 (phase IIb) formulated in IC31 as adjuvant and fusion proteins of Antigen 85B and TB10.4 (H4) or Antigen 85B, ESAT-6and Rv2660c (H56) (73, 112–114).ID93 (phase IIa) composed of GLA-SE as adjuvant and a fusion protein of 4 antigens, namely Rv2608, Rv3619, Rv3620 and Rv1813 (115, 116).M72 (phase IIb completed) composed of AS01E as adjuvant and a fusion protein of 2 antigens, Rv1196, and Rv0125. M72 has completed a phase IIbtrial revealing its partial protective efficacy (for further details see text) (65, 66, 117).

• Compositions of adjuvants:IC31, cationic peptides plus TLR-9 agonist;CAF01, cationic liposome vehicle plus immunomodulatory glycolipid;GLA-SE, Squalen oil-in-water emulsion plus TLR-4 agonist;AS01E, liposomes with monophosphoryl lipid A plus saponin QS21.

• Viable vaccines undergoing clinical testing are:MTBVAC (phase IIa completed), a genetically attenuated Mtb vaccine (118, 119).VPM1002 (several phase III trials), a rBCG vaccine (for further details see text) (84, 85).

• Killed whole cell vaccines include:DAR-901 (killed M. obuense) which had already completed a phase III trial under a different name (120–123) and is now under re-evaluation (phase Itrial completed) (124).MIP (phase III) based on killed M. indicus pranii organisms (125–127).M. vaccae (phase III) based on killed M. vaccae (128–132).RUTI (phase IIa) a purified killed vaccine of Mtb fragments (133–135).

• Therapeutic vaccines: The above vaccine trials assess outcome of preventive vaccination. Several candidates are also tested as therapeutic vaccineseither for TB treatment in adjunct to canonical chemotherapy or for PoR of TB patients who were cured from TB by canonical chemotherapy but mayundergo recurrence (136).Therapeutic vaccines in clinical trials include:H56:IC31 (phase I), a subunit protein formulation;ID93:GLA-SE (phase I), a subunit protein formulation;RUTI (phase IIa), a purified killed vaccine of Mtb fragments;TB-FLU-04L (phase IIa), a viral vectored vaccine;MIP (phase III completed), a killed M. indicus pranii preparation;M. vaccae (phase III completed), a killed M. vaccae preparation;VPM1002 (phase III), a live rBCG vaccine.

CURRENT STATUS OF TUBERCULOSISEPIDEMIOLOGY AND THETUBERCULOSIS VACCINE PIPELINE

According to the latest TB report of the World HealthOrganization (WHO), 10 million individuals developed activeTB disease and 1.5 million died of TB in 2018 (11). Globally1.7 million individuals are Mtb infected (LTBI, incipient TB,

subclinical TB) (52). Thus, the goal of the WHO to eliminateTB over the next decades requires much better interventionmeasures and notably a highly efficacious vaccine (10). BCGfails to protect against pulmonary TB, which is not only themost prevalent form of disease but also the major source oftransmission. This has led to several attempts to design novelvaccination regimens (18). Numerous vaccine candidates haveentered clinical trials and first promising results have been

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obtained (see below). Current vaccine candidates undergoingclinical testing are viral vectored vaccines expressing a few Mtbantigens, adjuvanted subunit vaccines typically comprising fusionproteins representing two to four Mtb antigens, killed whole cellvaccines and viable whole cell vaccines. Further details can befound in Box 1. The vaccine candidates are tested in differentclinical situations. These are:

(i) Prevention of Infection (PoI): This clinical endpoint canbe applied for pre-exposure vaccination, i.e. vaccinationof individuals who have not yet encountered Mtb. Themost important target group for PoI are neonates. TheWHO has prioritized a vaccine to lower the risk of Mtbinfection (11).

(ii) Prevention of Disease (PoD): It is obvious that PoI willresult in PoD. The major target population for PoD,however, are individuals with LTBI. Cutting the risk of TBdisease in individuals with LTBI has also been prioritizedby the WHO (11).

(iii) Prevention of Recurrence (PoR): In high endemic areas,ca. 10% of TB patients who had been cured bycanonical drug treatment undergo recurrence, either dueto reinfection or relapse (64).

(iv) Therapeutic Vaccination in Adjunct to Canonical DrugTreatment: Such a vaccination regimen gains increasingimportance for patients with multi or extensively drug-resistant TB (MDR / XDR-TB) (16). An estimated halfmillion of active TB patients suffer from MDR-TBand 50,000 to 100,000 individuals from XDR-TB (1).Vaccines for PoR are sometimes considered as therapeuticvaccines, as well.

This review will focus on vaccines that prevent active TB eitherthrough PoI, PoD, or PoR.

PREVENTION OF DISEASE BY THESUBUNIT VACCINE M72 IN A PHASE IIbCLINICAL TRIAL

The M72 vaccine candidate developed by GlaxoSmithKlinehas successfully completed a phase IIb clinical trial (65, 66).Participants of this study were HIV− adults with LTBI who hadbeen immunized with BCG as infants. Hence, the study was apost-exposure booster immunization with a subunit vaccine withPoD as clinical endpoint. The clinical endpoint was determinedafter 2 years of follow-up as pulmonary TB in absence ofHIV infection (66). The study revealed ca. 50% protection overplacebo control. The follow-up study confirmed the efficacy afterthe third year (65). This is the first vaccine trial to provideevidence for PoD in human TB. A positive control with BCG wasnot included in this study. It is hoped that global gene expressionprofiling and immunologic data will provide information aboutpotential mechanisms underlying PoD induced by this vaccine.The vaccine comprises two TB antigens formulated in a potentadjuvant, AS01E (see Box 1). This adjuvant had been developedas part of the adjuvant system (AS) series and is also used in the

shingles vaccines, Shingrix, and the malaria vaccine, Mosquirix(67). Availability of AS01E is limited and production cost is high. Ithas to be seen whether and how these limitations affect supply ofthis vaccine for broad-scale immunization programs. Satisfactorysupply of vaccines for poverty-related diseases including TB andmalaria strongly depends on an affordable price (68).

PROMISING PREVENTION OF DISEASEDATA IN NON-HUMAN PRIMATES (NHP)BY A VIRAL VECTORED TUBERCULOSISVACCINE CANDIDATE

Cytomegalovirus (CMV) based vaccines have been studied ina number of infectious diseases (69). Notably, in a simianimmunodeficiency virus (SIV) model of rhesus macaques, CMVvectored vaccines expressing SIV antigens have shown profoundprotection mediated by CD4 and CD8 T lymphocytes (69).These T cells have been characterized as effector TEM cells andtransitional effector memory T cells. Based on these findings, aTB vaccine candidate was designed which is based on a CMVvector expressing 6 or 9 Mtb antigens (70). This vector has beentested for PoD in rhesus macaques and was shown to induceprofound protection against TB disease (70). Importantly, in aproportion of animals, evidence for sterile eradication of Mtbby this CMV-vectored TB vaccine was obtained. As expected,the vaccine induced profound CD4 and CD8 T cell responses aswell as marked IFN-γ and TNF secretion. In contrast, antibodyresponses were not induced significantly. The protective CD8T cell population was not only restricted by MHC I, butalso by MHC-E or MHC II. BCG administered intradermallyalso induced protection, albeit weaker. Intriguingly, prime withBCG and boost with the CMV-based TB vaccine revertedthe strong protective effect of the CMV vaccine to levelsof protection induced by BCG. Gene expression profiling ofvaccinated animals indicated a role for neutrophils in protectioninduced by the CMV vectored TB vaccine. In conclusion, despitecertain disadvantages of CMV-vectored vaccines in general, theCMV-based TB vaccine represents a promising candidate whichdeserves further investigation. Obviously, the nullifying effectof BCG prime on protective efficacy induced by the CMVTB vaccine boost needs particular attention. Neonatal BCGimmunization is done routinely in high TB endemic areas as partof the expanded program of immunization (EPI) recommendedby WHO. Hence a new vaccine that provides no added value forBCG-immunized individuals will face major issues before it canbe further developed. Similarly, a recent study revealed that inNHP boosting BCG with M72 or H56 (see Box 1) vaccines failedto enhance protection induced by BCG (71).

RECENT FINDINGS WITH THECANONICAL BACILLECALMETTE-GUÉRIN VACCINE

Two recent studies on BCG immunization have revealedmarked impact of the vaccination regimen (72, 73). In the

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first study, NHP were immunized with BCG intravenously (72).Earlier research in the 1970s had already provided compellingevidence that intravenous immunization with live BCG inducessuperior protection against TB as compared to other routes ofadministration in NHP with evidence for sterile eradication ofMtb (74, 75). Thus, in one study 3/3 animals were markedlyprotected against TB as measured by hematogenous spread,lymphadenopathy and lung involvement (74). On the otherhand, profound splenomegaly was reported after intravenousadministration of live BCG. Probably this significant adverseevent was the major reason that such studies were not followedup. Only very recently this approach was investigated in greaterdepth. It was shown that intravenous immunization of NHPwith BCG induced more profound protection than intradermalor aerogenic vaccination (72). Indeed, from a proportion ofanimals receiving BCG by the intravenous route no Mtb couldbe recovered. This study also included a series of highlysophisticated immunologic and pathologic analyses. It was foundthat antigen responses of CD4 and CD8 T lymphocytes wereinduced substantially by intravenous immunization prior toMtb challenge, whereas γ/δ T cells and MAIT cells were,similarly, activated as in groups receiving other routes ofimmunization. The T cell response was mostly of TH1 typewith some contribution of TH17 type. On the negative side,splenomegaly was observed after intravenous immunizationwith a ca. twofold enlargement of spleens compared tocontrols. However, splenomegaly was transient and 6 monthsafter BCG immunization no differences were observed inspleen size across the different experimental groups includingintravenous administration. Six months after immunization,animals were challenged with a low dose of Mtb. Positronemission tomography – computed tomography (PET/CT) scansrevealed fewer granulomas in the intravenously immunizedanimals compared to controls. These findings provide proofof concept that BCG immunization can induce profound, insome cases sterile, protection in NHP. It needs to be seen howfar the splenomegaly observed will be prohibitive for clinicalstudies in humans.

The second recent study tested the outcome of BCGbooster vaccination in Mtb unexposed adults (73). Boostervaccination with BCG had been performed previously althoughgenerally it was not endorsed because of the potential risk ofadverse events. This assumption was largely based on anecdotalreports describing occasional adverse events after repeated BCGimmunization in individuals with LTBI and frequent severeevents in TB patients. Principally, BCG revaccination in Mtbuninfected individuals does not cause major side effects andin the recent formal clinical trial, BCG revaccination of Mtbunexposed individuals demonstrated partial prevention of stableMtb infection (73). More precisely, exposure was determinedindirectly via an IFN-γ release assay (IGRA) which determinesIFN-γ secretion by canonical T cells after in vitro restimulationwith Mtb specific antigens (76–78). This assay is mostly basedon CD4 T cell responses with some contribution of CD8 Tcells. Whilst initial IGRA conversion did not differ betweenBCG immunized and untreated study participants, sustainedIGRA conversion was significantly reduced by ca. 45% in BCG

immunized study participants over controls (73). These findingscan be interpreted to mean that stable Mtb infection is preventedby BCG revaccination although in fact it is based on reduced Tcell responses as measured by IGRA. It remains to be establishedmore precisely whether prevention of sustained IGRA conversiondirectly translates into long-term PoI and consequently PoD.Previous observational studies had evaluated PoD by BCGrevaccination based on epidemiologic data. Generally, theydid not find significant differences between controls and BCGrevaccinated individuals (79–81).

These two studies provide strong evidence that theoutcome of BCG vaccination is markedly influenced bythe kind of administration, notably route of immunization(intravenous) and type of vaccine schedule (pre-exposurerevaccination). In conclusion, the BCG vaccine still providesroom for improvement.

VPM1002

One of the most advanced TB vaccines, VPM1002, was improvedby genetic modification (82). VPM1002 is a recombinant BCG(rBCG) which expresses listeriolysin from Listeria monocytogenesand is devoid of urease C (83). Development of this vaccine hadstarted in the 1990s with the aim to improve BCG by endowingit with the capacity to stimulate a broader, more efficaciousT cell response.

VPM1002 has successfully completed phase I and phase IIaclinical trials proving its safety and immunogenicity in adults andneonates (84, 85). A phase II clinical trial in HIV exposed andunexposed neonates has been completed and awaits unblinding(NCT 02391415). A phase III clinical trial in HIV exposed andunexposed neonates is being prepared and expected to startin 2020. This trial has been designed as pre-exposure BCGreplacement for infants with PoI as clinical endpoint. In thisclinical trial, termed priMe, neonates will be immunized withVPM1002 or BCG as comparator at several sites in Sub-SaharanAfrica. A phase III clinical trial with VPM1002 assessing PoRis currently ongoing in India (NCT 03152903). For this trial,patients with TB who had been cured by drug treatment are beingrecruited. An estimated 10 % of these individuals will developactive TB disease due to reinfection or relapse within 1 year aftercompletion of drug treatment. The clinical trial therefore willreveal whether vaccination with VPM1002 given 3 months aftercompletion of drug treatment can prevent recurrence. A phaseIII household contact trial has been launched in July 2019 bythe Indian Council for Medical Research (ICMR), in whichVPM1002 and another vaccine candidate (MIP, see Box 1) willbe assessed for PoD in household contacts of patients withactive pulmonary TB disease. In addition, VPM1002 is also beingassessed as therapeutic agent against non-muscle invasive bladdercancer as substitute for BCG (NCT 02371447). The canonical TBvaccine BCG is the preferred immunomodulatory medicine fortreatment of bladder cancer and the current clinical trial assesseswhether VPM1002 is safer than and at least equally efficientas BCG against recurrence of bladder cancer. In conclusion, acentury after the introduction of the original vaccine BCG, there

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is hope for a revival of an improved BCG-based TB vaccine.A rationally revamped BCG could contribute to the solutionof the TB crisis.

HOW DOES THE INTRACELLULARBEHAVIOR DIFFER BETWEEN VPM1002,BACILLE CALMETTE-GUÉRIN, ANDMtb?

Both BCG and Mtb reside in phagosomes, which are arrested atan early stage by neutralization of the phagosomal pH to preventits acidification (21). Consequently, phagolysosome fusion isdiminished. Yet, BCG is degraded in the phagosome whereasMtb survives in phagocytes for prolonged periods of time. Onlyrecently, virulence mechanisms of Mtb absent from BCG havebeen elucidated. Although several sub-strains of BCG exist,it is now clear that the critical step which occurred duringattenuation of the parental Mycobacterium bovis strain wasthe loss of the region of difference (RD) 1 which encodes anumber of gene products mediated through the ESX/type VIIsecretion system and capable of perturbating the phagosomalmembrane (86). Membrane perturbation by the RD-1 geneproducts of Mtb leads to inflammasome activation, apoptosisand autophagy (Figure 2). The signaling cascades involve nod-like receptor protein 3 (NLRP-3) and absent in melanoma2 (AIM-2), responsible for IL-1 and IL-18 processing fromtheir respective precursor molecules by the inflammasome aswell as STING responsible for autophagy and type I IFNdependent responses (87). STING senses cyclic guanosinemonophosphate-adenosine monophosphate (cGAMP) derivedfrom double-stranded DNA of Mtb via the enzyme cyclicguanosine monophosphate-adenosine monophosphate synthase(cGAS). All these sequelae are caused by Mtb but notor less so by BCG.

For the design of VPM1002, BCG was equipped withlisteriolysin from L. monocytogenes which facilitates perturbationof the phagosomal membrane thereby inducing stronger Tcell responses (83). Listeriolysin is a thiol-activated perforin,which perforates cholesterol containing membranes at an acidicpH (88–90). This pH restriction generally prevents listeriolysinactivity in the extracellular milieu with neutral pH, e.g. blood andinterstitial space. It is however, achieved during natural infectionof phagocytes with L. monocytogenes which allows secretionof biologically active listeriolysin. Because BCG neutralizesthe phagosomal compartment, acidification is not achieved.For this reason, the urease C encoding gene was deletedin VPM1002 (83). This enzyme is responsible for ammoniaproduction and thereby participates in neutralization of thephagosome where BCG resides (21). Accordingly, VPM1002lacking urease C favors phagosomal acidification and therebysecretion of biologically active listeriolysin (Figure 2). Oncelisteriolysin has reached the cytosol, it is rapidly degraded. Thisis due to the amino acid sequence proline-glutamate-serine-threonine (PEST) in the listeriolysin amino acid sequence whichpromotes its ubiquitination (Figure 3) (88–90). This represents

an inbuilt safety mechanism, which restricts listeriolysin-activityto the perturbation of the membrane of the phagosome, whereVPM1002 resides and prevents further potentially detrimentaleffects on cell membranes. The RD-1 encoded machinery of Mtbis not endowed with such a safety mechanism.

Similar to the RD-1 machinery in Mtb, listeriolysin-mediatedperturbation of the phagosomal membrane by VPM1002 resultsin inflammasome activation through AIM-2 (91). Hence, IL-1and IL-18 are processed from their respective precursors.These proinflammatory cytokines create a milieu favorable foractivation of TH1 and TH17 cells. Listeriolysin also facilitatesautophagy via AIM-2 and STING by promoting sensing ofdouble-strand mycobacterial DNA derived from VPM1002 viacGAS and cGAMP after its egress into the cytosol (91).In addition, membrane perturbation by listeriolysin causesapoptosis, which leads to cross priming of T cells (92). Togetherthese mechanisms improve vaccine efficacy of VPM1002 ascompared to canonical BCG (82, 93). Moreover, VPM1002was shown to be safer than BCG in preclinical studies (83).In experimental models improved stimulation of both CD4and CD8 T cells has been demonstrated (92) as well as moreprofound activation of TH17 cells in addition to TH1 cells(94). In addition, central memory T cells were more stronglystimulated by VPM1002 as compared to canonical BCG (50).Finally, VPM1002 was shown to stimulate higher serum levelsof specific antibodies both in animal models and in human (50,84, 85). In conclusion, VPM1002 stimulates an immune responseof more depth and breadth and at the same time expresses lowervirulence as compared to BCG (82).

LEARNING FROM INDIVIDUALSRESISTANT TO STABLE Mtb INFECTIONAND THOSE CAPABLE OF ERADICATINGMtb AFTER STABLE INFECTION

Individuals with LTBI are generally identified by tuberculinskin test (TST) or IGRA (Figure 4) (76–78). Accordingly,identification of the 1.7 billion individuals on this globe withLTBI is based on measurements of T cell responses against Mtbantigens. These antigens are relatively undefined mixtures of Mtbproteins (purified protein derivative, PPD) in the case of TST andwell defined Mtb proteins and/or peptides in the case of IGRA.

Several lines of evidence suggest that a distinct populationof individuals remain Mtb uninfected despite their close andprolonged contact with patients with active pulmonary TB whocontinuously expel Mtb (95–98). This notion is based on thefinding that such individuals do not convert when tested byTST or IGRA. Assuming that the lack of the canonical immuneresponse determined by TST and/or IGRA reflects absence ofMtb infection, the following scenario arises (Figure 3): Initially,household contacts of a patient with active pulmonary TB fallinto two groups; those who are already LTBI because of previouscontact and hence are TST+/IGRA+, and naïve individuals whoare TST−/IGRA−. Due to the intensive contact, most of thenaïve individuals will convert to TST+/IGRA+ and most of them

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FIGURE 2 | Major mechanisms underlying induction of the host immune response by VPM1002 and M. tuberculosis (Mtb) (for further details see text). (A) VPM1002.VPM1002 (rBCG1ureC::Hly) expresses listeriolysin and lacks urease C activity. Following phagocytosis, VPM1002 ends up in a phagosome. Principally phagosomes

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FIGURE 2 | Continuedbecome acidic after uptake of particles, but BCG and Mtb actively keep the phagosomal pH neutral. Due to the absence of ureaseC in VPM1002, acidification takesplace. This facilitates perturbation of the phagosomal membrane by biologically active listeriolysin. (1) Membrane perturbation allows egress of antigens into thecytosol for processing through the MHC class I pathway. (2) Perturbation can lead to apoptosis. (3) Double-strand DNA released into the cytosol is sensed byabsence in melanoma 2 (AIM2). (4) AIM2 activates the inflammasome to generate IL-1β and IL-18. (5) Cyclic GMP-AMP synthase (cGAS) is formed which is thentransformed into cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). (6) The latter molecule is sensed by stimulator of IFN genes (STING) whichinduces autophagy and type I IFN responses. (7) Antigen egress into the cytosol allows stimulation of CD8 T cells in addition to CD4 T cells. (8) Apoptosis promotescrosspriming. (9) Autophagy accelerates elimination of VPM1002 and improves antigen presentation and T cell stimulation. (10) IL-1β and IL-18 induce aninflammatory response. Through these mechanisms, VPM1002 induces an immune response with more depth and breadth than parental BCG (B) Mtb. The genomeof Mtb comprises the region of difference 1 (RD-1) which encodes numerous virulence factors which are absent in BCG. Notably genes for Esx dependentmechanisms cause perturbation of phagosomal membranes, very similar to VPM1002. For further details see (A). Because the RD-1 encoded gene products are notdegraded after their egress into the cytosol, pathologic consequences prevail. Moreover, RD-1 encoded gene products are not controlled by pH. Hence, inbuiltsafety mechanisms of VPM1002 are absent from Mtb (see also Figure 3).

FIGURE 3 | Safety mechanisms of listeriolysin render VPM1002 less virulent than parental BCG. Listeriolysin contains a PEST-like sequence which promotes itsdegradation. (1) Only at acidic pH, listeriolysin is biologically active and hence perturbates the phagosomal membrane. (2) In the cytosol, monomeric listeriolysinaggregates. (3) Aggregated listeriolysin is degraded by ubiquitin resulting in inactive peptides. (4) Multimeric listeriolysin complexes are formed at the plasmamembrane. (5) These complexes are translocated into autophagosomes by ubiquitin. (6) These listeriolysin complexes are inactivated in the phagosome.PEST = Proline (P), Gutalate (E), Serine (S), and Threonine (T). Modified from (88–90).

remain TST+/IGRA+ over longer periods of time, if not lifelong.However, a small group may revert to TST−/IGRA− indicatingthat they are capable of eradicating Mtb before they becomepermanently infected. The recent BCG revaccination trial onPoI (73) described above did not reveal significant differencesbetween BCG-immunized and control groups in early conversionto IGRA+. Yet, a 45% reduction in sustained IGRA+ (determinedat later time points) was observed in the BCG immunizedgroup as compared to controls without BCG immunization.Moreover, observational studies have identified a distinct groupof permanent non-converters (TST−/IGRA−) generally in theorder of 20% (95–98).

Obviously, the described effects could also be due to technicalreasons and the TST−/IGRA− group could be infected with Mtbbut missed by TST/IGRA because these individuals develop aprotective immune response which is not detected by TST andIGRA. Underlying mechanisms could include antibodies, MAITcells, γδ T cells, NK cells and NKT cells (32, 36–45). Furthermore,it remains unclear whether all TST+/IGRA+ individuals areindeed Mtb infected or whether at least a subgroup has succeededin eliminating Mtb but remains TST+/IGRA+ because of astrong memory T cell response which persists in absence of Mtbantigens. Principally, immunology defines memory as a state ofimmunity in absence of nominal antigen(s).

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FIGURE 4 | Fate of household contacts of a TB index case. Household contacts of a TB index case are either already latently TB infected (LTBI) or do not showevidence for immunity against Mtb infection. After sustained contact with a TB index case, the majority of naïve individuals will rapidly convert to LTBI because they

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FIGURE 4 | Continuedmount an immune response against Mtb infection. Most of these early converters will remain LTBI and hence become sustained converters. A small proportion ofearly converters reverts to naïve, i.e. devoid of a measurable immune response to Mtb infection. Some naïve individuals will remain permanent non-converters, i.e.they do not change their status of absent immunity indicating absence of Mtb infection. Finally, some individuals with LTBI will revert to naïve, i.e. they lose theirdetectable immune response to Mtb indicating elimination of Mtb. The mechanisms underlying these conversions/reversions remain elusive. (A) Indicates responsein TST/IGRA and (B) depicts resulting conclusions on conversion/reversion (for further details see text).

Another interesting group may arise years after primaryMtb infection. Whilst many individuals with LTBI remainTST+/IGRA+ livelong, some individuals revert to TST−/IGRA−.It is likely that in these individuals, reversion from TST+/IGRA+to TST−/IGRA− reflects sterile eradication of Mtb. Yet, it cannotbe excluded formally that these individuals remain Mtb infectedand control infection by unknown immune mechanisms notdetected by TST/IGRA such as antibodies and unconventionalT cells. In any case, the permanent non-converters and thelate reverters are highly interesting study groups which providethe opportunity to gain deeper insights into the mechanismsof protection against Mtb. TB vaccines which prevent stableinfection with Mtb and thereby prevent LTBI and active TBdisease would be highly desirable. The specific mechanismsunderlying permanent non-conversion and late reversion couldbe elucidated by determining transcriptomic, metabolomicand immunologic markers and signatures which distinguishpermanent non-converters and late reverters from sustained andlivelong reverters, respectively (26, 99).

OUTLOOK AND FUTURE

Over the last decade, the TB vaccine pipeline has significantlyprogressed. First, a number of vaccines is ready for clinicalefficacy testing for PoI, PoD, or PoR (see Box 1). This impliesthat several vaccine candidates have already proven their safetyand immunogenicity. Second, several positive signals arose fromclinical trials over the last years including proof of conceptthat a subunit vaccine empowered by a strong adjuvant canpartially protect against active TB when given post-exposure toindividuals with LTBI (65, 66). Third, BCG revaccination ofMtb unexposed individuals has provided indirect evidence forpartial prevention of sustained Mtb infection (73). Obviously,major issues remain to be solved. These include: First, BCGrevaccination outcome was determined by IGRA which measurescanonical T-cell immune responses rather than Mtb infectionper se (see above). This raises the question whether BCGindeed prevented Mtb infection or whether infection occurredbut was controlled by alternative immune mechanisms suchas antibodies and/or unconventional T cells. Second, the M72clinical trial did not include BCG vaccination as a positivecontrol. Third, studies in NHP revealed that different subunitvaccine constructs including M72, H56, and the CMV vectoredTB vaccine failed to increase protection when given as boosteron BCG prime (70, 71). In sum, there is well justified hopefor better vaccines; but it remains difficult to predict whenand to which degree TB can be controlled by improvedvaccination strategies.

Nassim Nicholas Taleb is best known for describing the BlackSwan Concept which basically includes the notions (100): (i) rareand improbable events do occur more frequently than we assume;(ii) these extreme events can have enormous consequences;(iii) experts generally provide explanations post-hoc which werenot plausible ex-ante. This concept was aptly illustrated by thefinancial stock market crisis in 2008 when numerous stockowners who had gradually accumulated a financial depot wentbankrupt through a single event. The most illustrative descriptionfor the Black Swan concept is the life of a Thanksgiving Dayturkey which is taken care of very well over the first 1000 daysby feeding it with most nourishing food. An observer (includingthe turkey if it could do so) could conclude that the quality of lifeof this animal increases constantly. Yet, on day 1001, the butcherkills the animal unexpectedly in preparation for ThanksgivingDay. Obviously, this is an extreme event with a major impact onthe animal. This scenario can also be turned upside down into a

FIGURE 5 | Possible scenarios of TB vaccine development given thatadequate financial funding is provided for research & development (R&D).Upper, single step event; Lower, multistep event.

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positive direction, i.e. that an unexpected and improbable eventturns into something markedly better (an event which wouldperhaps be better described by the term Pink Swan). With respectto TB vaccine design, continuous funding into R&D (from basicresearch to preclinical and clinical development) will increaseour knowledge about the underlying mechanisms of protectionagainst TB and how this information can be harnessed for TBvaccine design (Figure 5).

For long periods of time research crawls, but every now andthen it jumps. By increasing funding, a fertile soil can be preparedfor R&D on better vaccines. Maybe this leads to an extremeevent (a single jump), resulting in a novel vaccine that fits allpurposes. More likely a couple of smaller, yet significant eventswill occur which ultimately lead to TB vaccines for differentpurposes. The type of vaccine and the time when it will be readyfor clinical licensing remain unclear as expected for a Black/PinkSwan. Yet, increased funding for R&D will favor chances ofsuccess. Undoubtedly, this cannot be accomplished free of cost;yet ultimately it will save cost by reducing the enormous expensescaused by the TB endemic. After all, annual cost for treatingactive TB disease globally has been estimated to be in the range

of 2 billion US dollars and the total burden of TB on the globaleconomy in the order of 100 billion US dollars (101).

It is worth to conclude with a citation from the resolutionadopted by the General Assembly of the United Nations from theHigh Level Meeting on the fight against TB in 2018 (1):

“Commit to mobilize sufficient and sustainable financing, with theaim of increasing overall global investments to 2 billion dollars,in order to close the estimated 1.3 billion dollar gap in fundingannually for tuberculosis research, ensuring that all countriescontribute appropriately to research and development . . ..”

AUTHOR CONTRIBUTIONS

SK conceived the idea and wrote the manuscript.

ACKNOWLEDGMENTS

The author thanks Diane Schad for superb graphics and SourayaSibaei for excellent help in preparation of the manuscript.

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Conflict of Interest: SK is co-inventor of the TB vaccine, VPM1002 and co-holderof a patent licensed to Vakzine Projekt Management GmbH, Hanover, Germanyand sublicensed to Serum Institute of India Pvt. Ltd., Pune, India. The vaccine iscurrently undergoing clinical trial testing.

Copyright © 2020 Kaufmann. This is an open-access article distributed under theterms of the Creative Commons Attribution License (CC BY). The use, distributionor reproduction in other forums is permitted, provided the original author(s) andthe copyright owner(s) are credited and that the original publication in this journalis cited, in accordance with accepted academic practice. No use, distribution orreproduction is permitted which does not comply with these terms.

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